1. Field of the Invention
The subject of the invention is a device and a procedure for isolating nucleic acids from the most different of starting materials containing nucleic acids, which both guarantees a very high quality of the isolated nucleic acids as well as allowing the isolation of quantitative yields.
2. Description of the Background Art
Under classical conditions the isolation of DNA from cells and tissues is carried out such that the starting materials containing the nucleic acids are digested under highly denaturing and reducing conditions, with in part the use of protein-degrading enzymes, the released nucleic acid fraction is purified in phenol/chloroform extraction stages and the nucleic acids are isolated by dialysis or ethanol precipitation from the aqueous phase (Sambrook, J., Fritsch, E. F. und Maniatis, T., 1989, CSH, “Molecular Cloning”).
These “classical procedures” for the isolation of nucleic acids from cells and especially from tissues are very time-consuming (in part longer than 48 h), require considerable apparative expenditure and moreover are not implementable under field conditions. In addition, such methods are hazardous to health, owing to the chemicals used in amounts that are not inconsiderable, such as phenol and chloroform.
Different alternative procedures for the isolation of nucleic acids from different biological starting materials allow the elaborate and health-damaging phenol/chloroform extraction of nucleic acids to be circumvented and a reduction in time expenditure to be achieved.
All of these procedures are based on a method for the preparative and analytical purification of DNA fragments from agarose gels developed and described for the first time by Vogelstein and Gillespie (Proc. Natl. Acad. Sci. USA, 1979, 76, 615 619). The method combines the dissociation in a saturated solution of a chaotropic salt (NaI) of the agarose containing the bands of the DNA to be isolated with binding of the DNA to glass particles. The DNA fixed to the glass particles is then washed with a wash solution (20 mM Tris HCl [pH 7.2]; 200 mM NaCl; 2 mM EDTA; 50% v/v ethanol) and then separated from the support particles.
Until now, this method has undergone a series of modifications and is currently used in different procedures for the extraction and purification of nucleic acids from different sources (Marko, M. A., Chipperfield, R. und Bimboim, H. G., 1982, Anal. Biochem., 121, 382 387).
In addition, a plurality of reagent systems exists world-wide today, predominantly for the purification of DNA fragments from agarose gels and for the isolation of plasmid DNA from bacterial lysates, and also for the isolation of longer-chain nucleic acids (genomic DNA, cellular total RNA) from blood, tissues or cell cultures.
All these commercially available kits are based on the well-known principle of binding nucleic acids to mineral supports in the presence of solutions of different chaotropic salts, and use suspensions of finely-milled glass powder (e.g. Glasmilk, BIO 101, La Jolla, Calif.), diatomaceous earths (Sigma) or silica gels as support materials (Diagen, DE 41 39 664 A1).
A procedure for the isolation of nucleic acids which is practicable for a number of different applications is proposed in U.S. Pat. No. 5,234,809 (Boom). A procedure is described therein for the isolation of nucleic acids from starting materials containing nucleic acids, whereby the starting material is incubated with a chaotropic buffer and a DNA-binding solid phase. The chaotropic buffer carries out both the lysis of the starting material as well as the binding of the nucleic acids to the solid phase. The procedure is well suited for the isolation of nucleic acids from small amounts of sample, and finds practical use particularly in the area of the isolation of viral nucleic acids.
Specific modifications of these procedures concern the use of novel support materials which have applicative advantages for particular problems (WO-A 95/34569).
More recent patent applications disclose that so-called antichaotropic salts can be used very efficiently and successfully as components of lysis/binding buffer systems for the adsorption of nucleic acids to silicate materials known and used by the person skilled in the art (EP 1135479).
Also part of the background art is the U.S. Pat. No. 6,207,445 B1, in which a simple to use filtration and extraction device for a biological fluid is described, which makes a sample directly available to an analytic procedure. The device is capable of making a purged fluid directly available for analysis or for disposal, for which it is suited to the specific analyte of note. And it is capable of capturing particle-shaped materials and allows for a further treatment, i.e. an extraction of these particles directly with the device. As soon as it has been extracted once, the device makes a fluid containing the analyte of note available to an analytic procedure. The device comprises a bendable body with an open upper and an inner wall which defines an inner chamber. A closing, or rather sealing, mechanism is set up in such a way to seal off the open upper end of the body. A graded filter apparatus containing at least one filter is incorporated into the body by a support apparatus. The elastic body is set up in such a way that, when pushed together by a user's finger, a positive pressure is generated in the chamber, which is enough to cause a fluid to flow into the chamber through the filter apparatus.
In U.S. Pat. No. 6,207,463, a device for separating magnetic particles from a stock composition is described. This device contains a longitudinally-stretched protective covering with an upper and a lower end, an inner space enclosed by the protective covering, which protrudes from its upper end to its lower end, an adjustable bar magnet which is arranged in the inner space, lengthwise to it.
A device for separating and cleaning a suspension with magnetic particles is described in the published international patent application WO2005/063831 A1. This device comprises a process area with mechanisms moving synchronously for the transport of magnetic microparticles in the x direction.
The analysis of the state of the art illustrates quite impressively that a plurality of possibilities exists to bind nucleic acids to solid support materials, in particular silicon-based mineral support materials, then to wash and to release once more the nucleic acids from the support material.
On the basis of the technologies described for the binding of nucleic acids onto solid support materials, there exists a plurality of commercially-available products which enable users to isolate nucleic acids from starting materials containing nucleic acids. Procedural solutions which are thereby increasingly interesting are those which permit nucleic acids to be automatically isolated and purified. This is accounted for by their reducing the considerable manual effort, or being able to implement high capacities of nucleic acid extraction. Taking account of these requirements, there are robot- or machine-based solutions for the isolation of nucleic acids. As a rule, this makes for very complex device configurations, which make the isolation of nucleic acids possible. In general, one can distinguish between two system configurations.
1. Automated procedures for the isolation of nucleic acids by the use of 96 Well filter plates to bind the nucleic acids.
2. Automated procedures for the isolation of nucleic acids by the use of magnetic particles to bind the nucleic acids.
The process of isolation of the nucleic acids is thereby implemented analogously to the manual procedure described. After lysis of the starting material takes place the binding of the nucleic acids to the particular solid support materials (filter plates or magnetic particles), and then the growing of the bound nucleic acids, drying of the support materials (removal of alcoholic components) and elution of the nucleic acids. The processing of the extraction in the case of use of filter plates is thereby carried out via vacuum or by means of pressure, or rather upon use of magnetic particles over a magnetic separation.
The task of the robot stations is, if possible, to automatically reproduce all the necessary procedural steps of a nucleic acid extraction. This is accomplished by complex pipetting steps by means of dispensation devices which must have the different buffer components to hand, vacuum stations for the filtration, or rather magnetic separators for use of magnetic particles, integration of complex centrifugal technology if appropriate. Heating units and shaking units are also preferably used to support the lysis processes.
However, there are only very few highly-specialized machines which enable the user to carry out the whole process of isolation of nucleic acids. In addition, these devices are often in no way universally applicable, but rather are only optimized for highly specialized application solutions. A further extreme disadvantage is the extremely high cost for the acquirement of such a machine. This is further exacerbated by what can be an extremely high rate of use of expendable materials, in particular pipette tips for the pipetting steps. Hence sometimes several hundred pipette tips can be required for certain uses for the isolation of nucleic acids by means of 96 Well filter plates.
Cost-effective systems are e.g. device solution which enable the user to isolate nucleic acids partially automatically by means of magnetic particles (e.g. KingFisher (trade name)). These devices have been specially developed for the isolation of nucleic acids and work according to a simple “walk-away” principle. The samples to be treated, as well as necessary buffer solutions for the binding, washing and elution steps, are carried over in reaction activities, such that the separation of the magnetic particles takes place over bar magnets which initiate the process of nucleic acid isolation by the contacting of the magnetic particles with the different buffer solutions. A new procedure is disclosed in EP1382675. Here, the process of isolation of nucleic acids is carried out by the use of a novel and extremely thin membrane. This membrane is a constituent of a filter cartridge. This filtration unit is a core part of an extraction device which carries out partial processes of the isolation of nucleic acids (binding, washing, elution) automatically. In this, a pre-lysed sample is introduced into the filtration unit by the user. The progress of the sample across the filter membrane can be carried out by vacuum. Subsequent wash solutions are automatically suspended and sucked across the membrane. The elution step takes place after addition of an elution buffer in the same form.
These latter device systems are much more cost-effective than the extraction robots already described.
But what is disadvantageous about these systems is the extremely small degree of automation. These systems only automate partial steps of procedures for the isolation of nucleic acids. The total progress of the sample pre-treatment, and in particular the sample lysis, is not carried out by means of these devices. The automated extraction is only carried out after lysis of the sample. In addition, the lysed sample is not once taken on automatically by the device, but rather must be manually introduced. The KingFisher system moreover requires time-consuming and labor-intensive steps in order to fill all buffer components into the reaction plastic intended for them.
The very low degree of automation therefore places a great limitation on the field of application of this device.
A further substantial problem in connection with an automation of procedures for the isolation and purification of nucleic acids, particularly those of complex starting materials (textiles, mousetail, plants, foodstuffs, stool samples etc.), consists in the separation of undissolved starting materials after lysis has been carried out. As a rule, up to now this could only be achieved by a centrifugation step for the pelleting of the undissolved components (further treatment with the supernatants), or rather by means of a centrifugation over a filter (further treatment with the filtrates). As the integration of such procedural steps is hardly possible with the established machines, automated purification of samples which are in this way problematic is impossible or only insufficiently achieved.